1. Field of the Invention
The present invention relates to a circular dichroism detector for HPLC.
2. Description of the Prior Art
As is well known, HPLC (High Performance Liquid Chromatography) is the most effective technique for carrying out separation analysis of optical isomers. Further, in such HPLC carried out in the prior art, the practical use of a polarimeter to detect optical rotation of a substance is used as a detection technique. Namely, optical rotation refers to the phenomenon in which the polarization plane of linearly polarized light passing through a substance is rotated by only a prescribed rotation angle. In this regard, it is possible to detect the properties of the substance by measuring such rotation angle.
However, because optical rotation is a phenomenon arising when the polarization direction of the linearly polarized light is rotated due to the circular birefringency (i.e., the difference in the indices of refraction for left-handed circularly polarized light and right-handed circularly polarized light) possessed by the optically active substance being analyzed, the base line fluctuation becomes large due to temperature changes of the polarizer utilizing birefringence, fluctuations in the index of refraction of the liquid phase inside the cell due to changes in the cell pressure, stresses exerted on the cell window, and depolarization due to the dispersion of bubbles and dust adhering to the cell window. For this reason, it is necessary to control ambient temperature changes within the range .+-.0.5.degree. C. Further, if the flow rate of the sample flowing through the inside of the cell changes, the base line will undergo large shift. Moreover, if the cell window is strongly fixed, it becomes impossible to measure the optical rotation.
On the other hand, one of the properties of optically active substances is circular dichroism. Namely, because the absorbance of a substance is different for left-handed circularly polarized light and right-handed circularly polarized light, measurements can be carried out based on the difference of such absorbances. In this way, by detecting the absorption difference, disturbance such as changes in the outside air temperature, pressure fluctuations inside the cell, and the adherence of bubbles, dust and the like to the cell window are canceled, thus making it possible to carry out stable measurements. For these reasons, it is also possible to detect optical activity under high pressure conditions.
In this connection, an example of a detector generally used in the prior art for measuring circular dichroism is shown in FIG. 1. As shown in this drawing, a Xe lamp is used as a light source 1, and light emitted from the light source 1 strikes an elliptical mirror M1 which changes the optical path to focus such light on an input slit S1. Next, the light which passes through the input slit S1 is sent to a double monochromator comprised of four mirrors M2.about.M5 and two reflection type birefringent prisms P1, P2 arranged in a prescribed positional relationship to disperse the light in the wavelength direction. Further, a slit S2 is arranged in the middle of this double monochromator. Then, because the final step mirror M5 focuses the light at the position of an output slit S3, all the linearly polarized light of a prescribed wavelength is outputted from the output slit S3.
The linearly polarized light outputted from the output slit S3 is passed through a final step PEM (Photo Elastic Modulator) 3. The light passing through this PEM 3 is changed from linearly polarized light to right-handed circularly polarized light to linearly polarized light to left-handed circularly polarized light, with elliptically polarized light having a slowly changing ellipticity angle existing between the linearly polarized light and the circularly polarized light. Further, compared with Faraday cells generally used in the prior art, a PEM has characteristics which make it possible to make the modulation angle extremely large. Accordingly, the PEM 3 alternately outputs left-handed circularly polarized light and right-handed circularly polarized light.
Next, this circularly polarized light is focused into a beam of light by a lens L1 and is focused by a lens L2 so as to be shone into a flow cell 5. A portion of this light is absorbed by a sample 6 flowing through the inside of the flow cell 5, with the remainder of such light being outputted by the flow cell 5. Then, the light outputted from the flow cell 5 is focused into a beam of light by a lens L3 in order to send such light to a photomultiplier serving as a light detector 7. Now, because the times when the light passing through the PEM 3 is right-handed circularly polarized and left-handed circularly polarized can be known by detecting modulation signals from the PEM 3, it is possible to determine the circular dichroism of the sample 6 from the difference in intensities of the light received for each type of circularly polarized light.
However, the prior art apparatus described above has various problems, as indicated below. First, because the above-described circular dichroism detector is used primarily to obtain a circular dichroism spectrum without being used exclusively for HPLC, the optical system must reduce stray light as much as possible and its wavelength resolution must be at or below 2 nm. Consequently, the overall size of such an apparatus becomes large, which in turn requires a large installation space and makes handling difficult. For example, one such apparatus has the dimensions 1250 mm (W).times.650 mm (D).times.405 mm (H) and weighs 100 Kg.
Furthermore, in the case where the optical system uses a birefringent prism, the small difference in the index of refraction of the prism for ordinary light and extra-ordinary light is used to separate the specified polarization light. As a result, in the prior art with double monochromator structure, the wavelengths only correspond up to 2 nm. When the slit width is enlarged beyond 2 nm, overlapping of ordinary light and extra-ordinary light occurs. Further, even at a slit width of 2 nm, the optical path length exceeds 3,000 mm. Thus, the dimensions stated above are established based on requirements related to structure and stray light control.
Further, because the prism dispersion is small for the near infrared component, the optical path length must be further extended in order to carry out measurements for such wavelength region, and this further enlarges the optical system, thus making the elements larger and related costs even higher.
Furthermore, in the case of a prism, because there are large changes in dispersion depending on wavelength, a variable slit width control is required, and because temperature changes occur easily, temperature adjustment and a thermally insulated structure are required. As a result, this leads to a complicated structure and higher costs.
Further, because such apparatus uses a double monochromator as an optical system which is effective in carrying out accurate circular dichroism spectrum measurements, the intensity of the light received by the light detector 7 is weak, thus making such apparatus inadequate for HPLC use in obtaining highly sensitive circular dichroism signals.
Furthermore, because the light received by such prior art apparatus is weak, a photomultiplier is used as the light detector 7. However, a photomultiplier is not only very expensive, but also large, thus interfering with miniaturization of the apparatus. Namely, in a very large optical system like that of the prior art, the occupancy ratio of the photomultiplier in the entire apparatus is not large, but in a case like the present invention where the structure of the optical system has been modified, with the installation area being reduced, the occupancy area of a photomultiplier can not be disregarded. Moreover, because the photomultiplier has a sealed construction containing a complex electrode structure inside a glass tube, warp stress remains on the light entrance window of the photomultiplier, and because this gives polarization characteristics to the light entrance window, such polarization characteristics will adversely affect the measurement results of the sample. Further, because the locality of the light-receiving area can easily receive effects such as magnetism and electromagnetic waves, various problems exist such as the need for adequate shielding. Further, because the light-receiving area is large, there is also the problem of it being easy for stray light to enter therein.